Blood-borne parasite infections present a major health problem in many areas of the world. Many of these areas lack both the equipment and skilled technicians to operate the equipment that are available for the detection of such parasites in a biological sample such as blood or a component thereof. In order to combat these problems, certain low-cost and low-skill level instruments have been developed but which provide accurate easily readable results. One such instrument comprises a capillary tube which contains a generally cylindrical mass having a specific gravity such that it will float in one of the cell layers when a blood sample is separated by centrifugation. The mass is selected such that it will form a thin annular space in the tube into which the parasite bearing cells will be crowded, thus increasing the concentration of parasites in a restricted area. The tube then is examined with the aid of a microscope for the presence of parasites within the annular region. U.S. Pat. No. 4,190,328 describes one such device employing this method. Commercially, the QBC.RTM. system (Becton Dickinson Primary Care Diagnostics) embodies this method.
A drawback to this method, however, is that absent the addition of a stain to highlight the presence of the parasite in any given cell the detection of such parasites may be difficult. Parasites may go through several different developmental stages in a particular host. Discriminating between stages is often difficult and requires a certain degree of skill and practice. The presence or absence of a particular stage may be indicative of the relative severity or stage of the infection.
In U.S. Pat. No. 4,190,328, acridine orange is disclosed as a membrane permeable stain that will stain the nucleic acids of parasites. Acridine orange, however, also is permeable in other blood cells and thus will stain to some degree nucleated white blood cells. Thus, where the clinician is not skilled in the identification of the stages of an infection, false positives may occur using a stain like acridine orange.
Another method for the analysis of blood borne parasites is not applicable to field use but is applicable to research use. This method comprises the use of a flow cytometer and a membrane permeable, nucleic acid stain such as thiazole orange. This method recently was described by Makler et al., Cytometry, 8:568 (1987).
Generally, this method comprises isolating a whole blood sample from a patient and staining the cells with thiazole orange. The stained cells then are run through the flow cytometer such as a FACScan.TM. (Becton Dickinson Immunocytometry Systems). As the cells pass through the flow cytometer, they pass through a sensing region, substantially one at a time, wherein each cell is scanned by light of excitation wavelength, typically light at 488nm from an argon laser. Light scattered by and fluorescent light emitted from each cell are detected by sensing means, such as photodetectors, and each cell is identified based upon all the light signals detected.
As noted in the reference, background staining of nucleated cells (both immature reds and all stages of whites) will occur as will staining of platelets. Although the staining of white blood cells can be "gated" out of the cell analysis, staining of the nucleated red cells and platelets cannot be gated out and thus will provide background fluorescence which may effect the identification of parasite bearing cells.
Accordingly, what is required for the improved practice of a method such as those described above is a stain that preferentially stains the nucleic acids of blood borne parasites with little or no staining of nucleated red and white blood cells and platelets.